This invention is in the field of microbiology. More specifically, this invention pertains to methods for the production of cyclic terpenoid compounds in microbial hosts that metabolize single carbon substrates as a sole carbon source.
Monoterpenes have value in the flavor and fragrance industries, as components in industrial solvents and in the pharmaceutical industry where selected compounds have shown promise as both chemopreventive and chemotheraputic agents for solid tumors.
Although found in a wide range of organisms, including bacteria, fungi, algae, insects and even higher animals such as alligators and beavers, monoterpenes are most widely produced by terrestrial plants such as components of flower scents, essential oils, and turpentine. One of the most common sources of the monoterpenes are the herbaceous plant and conifer turpentines. The pinene regioisomers (xcex1-pinene, xcex2-pinene) are 2 principal monoterpenes of turpentine as they serve as large volume aroma chemicals. Other essential oils (from orange, lime, lemon, and peppermint) are valued in flavoring and perfumery. The cyclization of linear terpenoid compounds to form cyclic derivatives may generate diverse aromatic structures with differing functionality.
At present the monoterpenes may be obtained either by extraction from natural sources or by chemical synthesis. Both processes are time consuming and expensive. Although small scale production of selected monoterpenes has been demonstrated in microbial hosts, a facile method for the production of monoterpenes on an industrial scale has yet to be reported. For example some monoterpene synthases have been successfully cloned and expressed in Escherichia coli. Specifically, limonene synthase, which catalyzes the cyclization of geranyldiphosphate to yield the olefin 4(S)-limonene in Perilla frutescens has been cloned into Escherichia coli and functionally expressed (Yuba et al. Arch Biochem Biophys 332:280-287, (1996)). Reports of microbial expression however have been limited to microbe traditionally used for fermentative production were grown on complex carbon substrates.
There are a number of microorganisms that utilize single carbon substrates as sole energy sources. These organisms are referred to as methylotrophs and herein as xe2x80x9cC1 metabolizersxe2x80x9d. These organisms are characterized by the ability to use carbon substrates lacking carbon to carbon bonds as a sole source of energy and biomass. A subset of methylotrophs are the methanotrophs which have the unique ability to utilize methane as a sole energy source. Although a large number of these organisms are known, few of these microbes have been successfully harnessed to industrial processes for the synthesis of materials. Although single carbon substrates are cost effective energy sources, difficulty in genetic manipulation of these microorganisms as well as a dearth of information about their genetic machinery has limited their use primarily to the synthesis of native products. For example the commercial applications of biotransformation of methane have historically fallen broadly into three categories: 1) Production of single cell protein, (Sharpe D. H. BioProtein Manufacture 1989. Ellis Horwood series in applied science and industrial technology. New York: Halstead Press.) (Villadsen, John, Recent Trends Chem. React. Eng., [Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East., New Delhi, India; Naguib, M., Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ. Arab Pet. Exporting Countries, Kuwait, Kuwait.); 2) epoxidation of alkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3) biodegradation of chlorinated pollutants (Tsien et al., Gas, Oil, Coal, Environ. Biotechnol. 2, [Pap. Int. IGT Symp. Gas, Oil, Coal, Environ. Biotechnol.], 2nd (1990), 83-104. Editor(s): Akin, Cavit; Smith, Jared. Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9633821; Merkley et al., Biorem. Recalcitrant Org., [Pap. Int. In Situ On-Site Bioreclam. Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson, Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio: Meyer et al., Microb. Releases (1993), 2(1), 11-22). Even here, the commercial success of the methane biotransformation has been limited to epoxidation of alkenes due to low product yields, toxicity of products and the large amount of cell mass required to generate product associated with the process.
One of the most common classes of single carbon metabolizers are the methanotrophs. Methanotrophic bacteria are defined by their ability to use methane as a sole source of carbon and energy. Methane monooxygenase is the enzyme required for the primary step in methane activation and the product of this reaction is methanol (Murrell et al., Arch. Microbiol. (2000), 173(5-6), 325-332). This reaction occurs at ambient temperature and pressures whereas chemical transformation of methane to methanol requires temperatures of hundreds of degrees and high pressure (Grigoryan, E. A., Kinet. Catal. (1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No. 5,750,821). It is this ability to transform methane under ambient conditions along with the abundance of methane that makes the biotransformation of methane a potentially unique and valuable process.
Many methanotrophs contain an inherent isoprenoid pathway which enables these organisms to synthesize other non-endogenous isoprenoid compounds. Since methanotrophs can use one carbon substrate (methane or methanol) as an energy source, it is possible to produce monoterpenes at low cost. Furthermore, during the fermentation, volatile compounds can be easily removed as methane is passed through fermentation media. It is also advantageous to produce via bio-route since many of monoterpenes have chirality and it is difficult to control the synthesis and purification of specific chirally active compound in chemical synthesis.
A need exists therefore for a method of production of highly valuable monoterpenes from an inexpensive feedstock. Applicants have solved the stated problem by providing a C1 metabolizing microorganism having transformed with a gene encoding a cyclic terpene synthase, having the ability to produce to a variety of monoterpenes.
The invention provides a method for the production of a monoterpene comprising:
a) providing a transformed C1 metabolizing host cell comprising:
(i) suitable levels of geranyl pyrophosphate; and
(ii) at least one isolated nucleic acid molecule encoding a cyclic terpene synthase under the control of suitable regulatory sequences;
(b) contacting the host cell of step (a) under suitable growth conditions with an effective amount of a C1 carbon substrate whereby a monoterpene compound is produced.
Preferred single carbon substrates of the present invention include but are not limited to methane, methanol, formaldehyde, formic acid, methylated amines, methylated thiols, and carbon dioxide.
Preferred C1 metabolizers or facultative methylotrophs where obligate methanotrophic bacteria are most preferred. Most preferred C1 metabolizers are those obligate methanotrophs comprising a functional Embden-Meyerof carbon pathway, said pathway comprising a gene encoding a pyrophosphate dependent phosphofructokinase enzyme.
Preferred cyclic terpene synthases of the invention include but are not limited to limonene synthase, pinene synthase, bornyl synthase, phellandrene synthase, cineole synthase, and sabinene synthase.
In an alternate embodiment the invention provides for the expression of upper pathway isoprenoid genes for the donwstream produciton of monoterpenes, the upper pathway isoprenoid genes selected from the group consisting of D-1-deoxyxylulose-5-phosphate synthase (DXS); D-1-deoxyxylulose-5-phosphate reductoisomerase (DXR); 2C-methyl-d-erythritol cytidylyltransferase (IspD), 4-diphosphocytidyl-2-C-methylerythritol kinase (IspE), 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF), CTP synthase (IspA) and Geranyltranstransferase (PyrG).